CN110862979B - Mutant of alkaline protease and application thereof - Google Patents
Mutant of alkaline protease and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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Abstract
The alkaline protease mutant disclosed herein has an amino acid Ile-to-Ala transition at position 113 or further an amino acid Ile-to-Glu transition at position 140, relative to the original amino acid sequence. Recombinant bacillus subtilis for expressing the mutant is respectively constructed by encoding genes of the mutant, the enzyme activities of fermentation supernatant under the condition of pH7.0 are 5673U/mL and 6733U/mL respectively, and the enzyme activities are respectively improved by 130 percent and 154 percent compared with the enzyme activities of the bacillus subtilis for expressing wild alkaline protease subC.
Description
Technical Field
The invention belongs to the field of biotechnology and protein engineering, and particularly relates to an alkaline protease mutant protein.
Background
The alkaline protease (alkaline protease) is an enzyme capable of hydrolyzing peptide bonds of proteins under alkaline conditions (the pH is within the range of 9-11), the main component of the alkaline protease is endoprotease, the catalytic site is serine, and the alkaline protease is widely applied to industries such as detergents, foods, medical treatment, brewing, silk and leather making. The alkaline protease adopted at present is a proteolytic enzyme which is cultured by a bacterial protoplast mutagenesis method and is derived from bacillus subtilis 2709 and is prepared by deep fermentation, extraction and refining, belongs to serine alkaline protease, can hydrolyze peptide chains of protein molecules to generate polypeptide or amino acid, and has stronger capability of decomposing protein. The production process adopts the advanced technologies of microfiltration and ultrafiltration membrane separation, spray drying or vacuum freeze drying and the like. The alkaline protease is a popular washing additive in the current market, can greatly improve the washing and dirt removing capacity, particularly has unique washing effect on protein dirt such as blood stains, sweat stains, milk stains, oil stains and the like, is an enzyme occupying the largest proportion in industrial enzymes, and accounts for about 60 percent of the total annual sales volume all over the world. In production, the quality of the granular alkaline protease for the powder detergent is required to be dust-free, safe and sanitary; the compatibility with a detergent is good; good stability in detergents, etc. Therefore, there is a need to develop alkaline proteases with superior performance and better applicability.
Site-directed mutagenesis is the introduction of desired changes (usually changes that characterize favorable orientations) including base additions, deletions, point mutations, and the like, by Polymerase Chain Reaction (PCR) or the like, into a DNA fragment (which may be a genome or a plasmid) encoding a protein of interest. The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work. The in vitro site-directed mutagenesis technology is an important experimental means in the research of various fields of biology and medicine at present, is a convenient scheme for modifying and optimizing genes, and is a powerful tool for researching the complex relationship between the structure and the function of protein. The corresponding amino acid sequence and protein structure can be changed by site-directed alteration, deletion or insertion of specific base of a known gene, and the research of the expression product of mutant gene is helpful for human to understand the relationship between protein structure and function and investigate the structure/structural domain of protein. In recent years, the application of site-directed mutagenesis technology of enzyme mainly focuses on the aspects of improving the catalytic activity of enzyme, improving the substrate specificity, improving the thermal stability, enantioselectivity and the like. The site-directed mutagenesis technology of the enzyme opens up a novel approach for the structure and the function of the enzyme. For example, studies have been made to improve the cleaning performance of alkaline proteases by mutating the alkaline proteases to improve their specific activities, stability, etc. (Japanese patent laid-open No. 2010-273673 and Japanese patent laid-open No. 5202690). For example, CN109312323A discloses that mutants of alkaline proteases are obtained by substituting threonine for the amino acid residue at position 294 in the amino acid sequence of a parent alkaline protease, thereby improving the specific activity. CN105176951A based on the alkaline protease aprE from Bacillus clausii, three mutants were obtained by mutation, with 42%, 180% and 130% higher activity than the parent.
Therefore, when a specific protein is mutated, the selection of the mutation site and the determination of the mutation direction are often difficult to predict or unpredictable in advance, and particularly in the case of alkaline proteases, the prior art has limited knowledge of the relationship between the structure and the function of the enzyme, and therefore, a protein mutant having a desired functional property cannot be obtained by theoretical analysis in advance, and it is necessary to search in advance in order to obtain a mutation in a desired direction of performance.
Disclosure of Invention
The present invention has been made in an intensive study on alkaline protease, and finally, by finding out a suitable mutation site and a specific substituted amino acid, a mutant produced by Bacillus licheniformis (B.)Bacillus licheniformis) Starting from the mature peptide of the alkaline protease gene subC, the alkaline protease mutant with obviously improved enzyme activity is finally obtained.
On the basis of the alkaline protease with a parent amino acid sequence of SEQ ID NO. 1, the 113 th amino acid of the alkaline protease is changed from Ile to Ala, and the amino acid sequence of the alkaline protease mutant is SEQ ID NO. 2.
Further, the present invention provides an alkaline protease mutant, which is an alkaline protease having the parent amino acid sequence of SEQ ID NO. 1 in which amino acid position 113 is changed from Ile to Ala and amino acid position 140 is changed from Ile to Glu (i.e., an alkaline protease having the amino acid sequence of SEQ ID NO. 2 in which amino acid position 140 is further changed from Ile to Glu), and which has the amino acid sequence of SEQ ID NO. 3.
The invention also provides an alkaline protease mutant coding gene, and the amino acid sequence of the coded polypeptide is shown as SEQ ID NO. 2 or SEQ ID NO. 3.
Further, the present invention provides a recombinant expression vector carrying the gene encoding the above alkaline protease mutant.
Still further, the present invention provides a recombinant host cell transformed/transfected with the recombinant expression vector described above. Preferably, the recombinant host cell is Bacillus subtilis.
In still another aspect, the invention also provides the use of the alkaline protease mutant as an additive for detergents. Further, the present invention provides a detergent composition comprising the above alkaline protease mutant. The lotion composition may be a powder detergent composition, but is preferably a liquid detergent composition. Wherein the alkaline protease mutant is added into the detergent composition in a form of fermented crude enzyme solution or purified.
The invention is based on the alkaline protease subC from bacillus licheniformis, obtains two alkaline protease mutants subC 1 and subC M2 by a site-directed mutagenesis technology, and ferments the mutants in bacillus subtilis, wherein the enzyme activities of the fermentation supernatant are 5673U/mL and 6733U/mL respectively, which are 130% and 154% respectively higher than the enzyme activity of bacillus subtilis (4356U/mL) expressing wild alkaline protease subC, and the invention has wider application.
Drawings
FIG. 1: a plasmid map of the alkaline protease expression vector;
FIG. 2: bar chart of relative enzyme activity of alkaline protease mutants.
Detailed Description
The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope of the invention.
The experimental techniques and experimental procedures used in this example are conventional techniques, unless otherwise specified, such as the conditions described in molecular cloning guidelines written by J. Sambruka (Sambrook), et al, or the procedures recommended by the manufacturer. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.
The terms and associated assay methods referred to in the present invention are explained below:
(1) the protease activity determination method comprises the following steps: adopts the method for determining the protease preparation of the national standard of the people's republic of China (GB/T25327-2009).
(2) Definition of enzyme activity unit: 1g of solid enzyme powder (or 1mL of liquid enzyme) hydrolyzes casein for 1min under the conditions of certain temperature and pH value to generate 1 mu g of tyrosine, namely 1 enzyme activity unit expressed by U/g (U/mL).
(3) Alkaline protease the activity of the protease was determined using the forskolin method using a solution comprising: folin use solution (one commercial Folin solution was mixed with two portions of water, shaken up), sodium carbonate solution (42.4g/L), trichloroacetic acid (65.4g/L), gradient pH buffer, casein solution (10.0 g/L). The reaction process is as follows: adding 1mL enzyme solution into the test tube, performing warm bath at 40 deg.C for 2min, adding 1mL casein solution, shaking, performing warm bath at 40 deg.C for 10min, adding 2mL trichloroacetic acid solution, and shaking (adding trichloroacetic acid and casein solution into blank control). Taking out and standing for 10min, and filtering with slow qualitative filter paper. Taking 1mL of filtrate, adding 5mL of sodium carbonate solution, adding 1mL of forskolin reagent solution, developing at 40 ℃ for 20min, and measuring absorbance at 680nm wavelength by using a 10mm cuvette.
(4) The identification of the alkaline protease mutant refers to the amino acid mutated in the alkaline protease mutant by the "amino acid substituted at the original amino acid position". Such as Ile113Ala, the amino acid at position 113 is replaced by Ile of the parent alkaline protease to Ala, and the numbering of the positions corresponds to that of SEQ ID NO 1 of the appendix sequence Listing. Such as Ile113Ala/Ile140Glu, indicating that both amino acids at position 113 and 140 have been mutated.
Example 1 construction of alkaline protease subcoC expression vector and recombinant Strain
The vector backbone sequence to be cloned was obtained by PCR amplification of the vector DNA sequence with the pMA5 plasmid as template, through the vector upstream primer 5'-gccttggcgcaagtagctagcgaacctctcattaaagcggacaa-3' (SEQ ID NO: 4) and the vector downstream primer 5'-ttgtccgctttaaagctagcatatcgaagtgagggcgccaaggc-3' (SEQ ID NO: 5). The nucleotide sequence of the complete gene of subbC (GenBank: X03341.1) is amplified by PCR through a fragment upstream primer 5'-agctatgcgatgctaggcatgatgcgcaaaaaaagcttctg-3' (SEQ ID NO: 6) and a fragment downstream primer 5'-gtgacgatggtcagctgtagctgggcggcagcttcaacgt-3' (SEQ ID NO: 7) by taking the genome DNA of the bacillus licheniformis as a template, so that the sequence of the alkaline protease carrying a signal peptide and a mature peptide is obtained, and the coded amino acid sequence of the alkaline protease is SEQ ID NO: 1.
the PCR amplification conditions are as follows: 10min at 94 ℃; 60s at 94 ℃, 60s at 58 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃. The PCR amplification product was recovered using the E.Z.N.A.gel Extraction Kit. And performing overlap extension PCR on the recovered enzyme gene sequence and the vector skeleton sequence to form a polymer, wherein an amplification system comprises the following steps: 5 XPHUSION HFBuffer 10 uL, 2.5mM dNTPs 8 uL, gene fragment (subcoC fragment) 4 uL, vector skeleton fragment (pMA5)6 uL, Phusion DNA Polymerase 1 uL, ddH2O21. mu.L. The amplification condition is 98 ℃ for 10 min; 10s at 98 ℃, 3min at 72 ℃ and 20 cycles; 10s at 98 ℃, 6min at 72 ℃ and 15 cycles; 10min at 72 ℃. Converting multimersBacillus subtilis1A751 host strain, screening positive transformant to obtain recombinant strain expressing wild type alkaline protease subbC, and naming the recombinant strain as Bacillus subtilis subbC. A plasmid in the Bacillus subtilis subcoC is extracted and named as pMA 5-subcoC (containing a wild-type subcoC gene), and the plasmid map of the plasmid is shown in figure 1.
The Bacillus subtilis 1A751 is transformed by a competent method, and the specific transformation process is as follows: will be freshly activatedB. subtilis1A751 was plated on LB (tryptone 1%, yeast powder 0.5%, NaCl 1%) plates to 5ml of GM I (GM I prepared by 1X minimum salt solution 95.6ml, 20% glucose 2.5ml, 5% hydrolyzed casein 0.4ml, 10% yeast powder 1ml, wherein 1X minimum salt solution was prepared by K2HPO414g/L，KH2PO46g/L，(NH4)2SO42g/L, trisodium citrate 1g/L, MgSO4·7H2O0.2 g/L, the solution was dissolved in distilled water successively, and cultured overnight at 30 ℃ with shaking at 125 rpm. The next day, 2ml of the suspension was transferred to 18ml of GM I and cultured at 37 ℃ and 250rpm for 3.5 hours. Then 10ml of the culture medium from the previous step was transferred to 90ml of GM II (GMII preparation method: 96.98ml of 1 × minimum salt solution, 2.5ml of 20% glucose, 0.08ml of 5% hydrolyzed casein, 0.04ml of 10% yeast powder juice, 1M MgCl20.25ml，1M CaCl2In the 0.05ml of the mixture,culturing at 37 deg.C and 125rpm for 90min, and centrifuging at 5000g for 10min to collect thallus. And (3) lightly suspending the thalli by using 10ml of original culture solution supernatant, wherein the suspended thalli are competent cells. Then, an appropriate amount of DNA was added to 0.5ml of the competence, and the mixture was subjected to shaking culture at 37 ℃ and 200rpm for 30min, followed by plating, further overnight culture at 37 ℃ and examination and verification of transformants the next day.
Example 2 construction of alkaline protease mutants Using Point mutation technique
Through the evolutionary analysis of the basic protease subC gene from the bacillus licheniformis, the amino acid sequence of the basic protease is compared with the amino acid sequences of other basic proteases with higher homology, and potential amino acid mutation sites with improved enzymological properties are searched. In particular to a method for searching mutation sites by selecting a ClustalX comparison method. ClustalX is a Windows version of the Clustal multiple sequence alignment program. Clustal X can provide an overall environment for performing multiple sequence and contour alignments and analysis results, and a multicolor mode can be adopted to highlight the characteristics of a conserved area in the alignment. The inventor selects dozens of alkaline protease amino acid sequences from bacteria, introduces the alkaline protease amino acid sequences into ClustalX software, utilizes a multiple sequence comparison tool to carry out dynamic sequence comparison, and adopts an ESPript method to carry out coloring enhancement. And analyzing the generated comparison result, selecting an amino acid site with poor sequence conservation of a plurality of alkaline proteases in evolution, namely high mutation occurrence frequency for mutation, and finally selecting amino acid residues of 113 th site and 140 th site for mutation by combining the candidate mutation site with three-dimensional structure auxiliary analysis of a PDB database.
A nucleotide mutation is introduced into the basic protease subcoC gene from the bacillus licheniformis by utilizing a point mutation technology. The first mutation results in a mutation of the encoded polypeptide at the Ile113Ala site (SEQ ID NO: 2). The PCR amplification system is as follows: 5 XPPhusion HF Buffer 10 uL, 2.5mM dNTPs 8 uL, template DNA pMA 5-subcoC plasmid (100 ng/. mu.L) 1 uL, Phusion DNA Polymerase 1 uL, ddH2O28. mu.L, upstream mutation primer 5'-gccttggcgcaaaccgttccttacggcgaacctctcattaaagcggacaa-3' (SEQ ID NO: 8), downstream mutation primer 5 ' -ttgtccgctttaatgagaggttcgccgtaaggaacggtttgcgccaaggc-3' (SEQ ID NO: 9). The reaction conditions are 98 ℃ for 10min, 98 ℃ for 10s, 55 ℃ for 20s, 72 ℃ for 3min, 20 cycles, and 72 ℃ for 10 min. the PCR product is transformed into Escherichia coli DH5 α competent cells, after a positive transformant is obtained, the Plasmid is extracted by E.Z.N.A. Plasmid Extraction Kit, and the Plasmid is named as pMA5-subCM 1.
The second mutation further mutates the encoded polypeptide at Ile140Glu site, i.e., the mutation comprises Ile113Ala/Ile140Glu overlapping site. The PCR amplification system is as follows: 5 XPPhusion HF Buffer 10 uL, 2.5mM dNTPs 8 uL, template DNA pMA 5-subcocMM 1 plasmid (100 ng/. mu.L) 1 uL, Phusion DNA Polymerase 1 uL, ddH2O28. mu.L, upstream mutation primer 5'-gccgtcctggatacaggagaacaagcttctcatccggactt-3' (SEQ ID NO: 10), downstream mutation primer 5'-aagtccggatgagaagcttgttctcctgtatccaggacggc-3' (SEQ ID NO: 11) under the conditions of 98 ℃ 10min, 98 ℃ 10s, 55 ℃ 20s, 72 ℃ 3min, 20 cycles, 72 ℃ 10min, transformation of the PCR product into E.coli DH5 α competent cells, Extraction of plasmids with E.Z.N.A.plasmid Extraction Kit after obtaining positive transformants, named pMA 5-subbCM 2, transformation of plasmids pMA 5-subbCM 1 and pMA 5-subbCM 2Bacillus subtilis1A751 host strain, screening positive transformants to obtain recombinant strains expressing wild type alkaline protease subcoC mutants, and respectively naming the recombinant strains as Bacillus subtilis subcoC 1(Bacillus subtilis subcoC 1) and subcoC 2(Bacillus subtilis subcoC 2).
EXAMPLE 3 evaluation of alkaline protease mutants
1. Shake flask fermentation
The 2 alkaline protease mutant recombinant strains (subBM 1 and subBM 2) and the control strain Bacillus subtilis subbC constructed above were inoculated into 50mL seed media (yeast extract 0.5%, tryptone 0.5%, glucose 1%, K)2HPO41.8%, kanamycin 25. mu.g/mL), at 34 ℃ for 8h with shaking at 210 rpm. Then inoculating 2.5mL of fermentation liquor into 50mL of fermentation medium (1-2% of yeast powder, 2-5% of bean cake powder, 5-10% of maltodextrin, 0.1-0.5% of sodium citrate, CaCl)20.1～0.5％，MgSO40.1～0.5％，K2HPO40.5-2%), at 34 ℃ and 250rpm for 72 h; centrifuging and taking supernatant.
2. Enzyme activity assay
The enzyme activities of the alkaline proteases of the 2 recombinant strains (subBM 1 and subBM 2) and the control bacillus subtilis subbC fermentation supernatant are respectively detected by adopting a national standard protease preparation determination method (GB/T25327-.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> alkaline protease mutant and use thereof
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<400>8
gccttggcgc aaaccgttcc ttacggcgaa cctctcatta aagcggacaa 50
<210>9
<211>50
<212>DNA
<213> Artificial sequence ()
<400>9
ttgtccgctt taatgagagg ttcgccgtaa ggaacggttt gcgccaaggc 50
<210>10
<211>41
<212>DNA
<213> Artificial sequence ()
<400>10
gccgtcctgg atacaggaga acaagcttct catccggact t 41
<210>11
<211>41
<212>DNA
<213> Artificial sequence ()
<400>11
aagtccggat gagaagcttg ttctcctgta tccaggacgg c 41
Claims (9)
1. An alkaline protease mutant is characterized in that the amino acid sequence of the alkaline protease mutant is shown as SEQ ID NO. 2 or SEQ ID NO. 3.
2. The gene encoding the alkaline protease mutant according to claim 1.
3. A recombinant expression vector carrying a gene encoding the alkaline protease mutant of claim 1.
4. A recombinant host cell transformed/transfected with the recombinant expression vector of claim 3.
5. The recombinant host cell of claim 4, wherein the host cell is Bacillus subtilis.
6. Use of the alkaline protease mutant according to claim 1 as an additive for detergents.
7. A detergent composition comprising the alkaline protease mutant according to claim 1.
8. A detergent composition as claimed in claim 7, which is a liquid composition.
9. The detergent composition of claim 7 or 8, wherein the alkaline protease mutant is added to the detergent composition in a fermented crude enzyme solution, or in a purified form.
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| JP7489134B2 (en) | 2021-12-02 | 2024-05-23 | 江南大学 | D-psicose 3-epimerase producing strain and use thereof |
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| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
| CN112574978B (en) * | 2021-01-19 | 2021-08-17 | 青岛尚德生物技术有限公司 | Protease mutant capable of improving alcohol-soluble protein degradation capacity and coding gene and application thereof |
| CN112662654A (en) * | 2021-01-28 | 2021-04-16 | 天津科技大学 | Alkaline protease mutant and application thereof |
| CN112725316B (en) * | 2021-03-04 | 2022-09-06 | 宁夏夏盛实业集团有限公司 | Alkallikrein 2018 mutant and preparation method thereof |
| CN116486903B (en) * | 2023-04-17 | 2023-12-29 | 深圳新锐基因科技有限公司 | Method and device for improving protein stability based on combination of homologous protein sequence evolution direction and free energy change |
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| EP2340306B1 (en) * | 2008-10-21 | 2016-12-14 | Basf Se | Nucleic acids with enhanced expression characteristics |
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| CN1505679A (en) * | 2001-05-02 | 2004-06-16 | �����ɷ� | Alkaline protease variants, and detergents and cleaning agents containing the same |
| EP2340306B1 (en) * | 2008-10-21 | 2016-12-14 | Basf Se | Nucleic acids with enhanced expression characteristics |
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| JP7489134B2 (en) | 2021-12-02 | 2024-05-23 | 江南大学 | D-psicose 3-epimerase producing strain and use thereof |
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